Glycogen in liver may be synthesized either directly from glucose or indirectly from lactate and alanine, which are cleavage products of glucose in nonhepatic tissues and which are returned to liver. We are developing methods to measure the contribution of these pathways to glycogen in the intact animal (rat) by using radioactive and nonradioactive tracers. The radioactive tracer used here is water labeled with tritium (3HOH). The incorporation of labeled hydrogen on carbons 2 and 6 of glucose provides an estimate of these pathways that is much more reliable than with 14C labeled substrates. Another method to measure the pathways of glycogen is by using nonradioactive carbon (13C). Glucose labeled uniformly with 13C (U-13C) will be infused intragastrically in rats. Liver glycogen will be isolated and assayed by gas liquid-mass spectroscopy to obtain the distribution of masses from 181 to 186 (1 to 6 labeled carbons per molecule). The ratio of glucose of mass 186 to that of all labeled molecules provides an estimation of the direct pathway. This method also does not depend on the specific activity of precursors and attainment of steady state. The use of labeled substrates and mass spectroscopy to study glycogen and glucose synthesis and the operation of the Krebs cycle will be explored. The value of the use of nonradioactive tracers such as 13C is that ultimately these procedures can be applied to humans without risks due to radioaction damage.